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分子分型在医院感染控制
中的作用
(the Role of Molecular
Typing in Nosocomial
Infection Control)
瑞金医院临床微生物科
瑞金医院医院感染办公室
杨 莉
分子分型(分子流行病学研究)
从核酸分子水平上分析医院感染的发生、发展规律及机理,
更加准确有效地进行医院感染管理控制,已成为当前国际
医院感染管理研究中的重要方向。
从患者分离株到病区周围环境株的比较分析;
从外源性感染到内源性感染
从某一医院的医院感染暴发到大范围甚至世界
范围的感染菌株流行变迁;
基因多态性分析技术已成为医院感染监测控
制的高水平研究领域。
2
微生物室在医院感染中的作用
accurately identifying
nosocomial
pathogens
detecting unexpected
antimicrobial-drug
resistance
epidemiologic typing
鉴定
特殊耐药菌的
检出
流行病学分型
Pfaller MA et al. The clinical microbiology laboratory and infection
control: emerging pathogens, antimicrobial resistance, and new
technology. Clin Infect Dis 1997;25:858-70.
3
分子分型
基因分型
分子流行病学
研究
PFGE(脉冲场凝胶电
泳)
RAPD(随机扩增DNA
多态性)
REA(限制性酶切)
ribotyping (核糖体分
型)
4
1、脉冲场凝胶电泳(PFGE)
制
备
琼
脂
糖
栓
块
消
酶
化
切
脉
冲
电
泳
结
果
解
释
5
PFGE原理
消化、释放出DNA
6
PFGE原理
Enzyme
酶切
约5~20个、长度
10~800kb的大片段
DNA
7
8
Tenover FC.et al.J Clin Microbiol,1995;33(9):2233~2239
PFGE原理
脉冲电泳
9
原理:
常规凝胶电泳
DNA
脉冲场凝胶电泳
10
PFGE原理
染色、拍照
时间消耗
从分离菌株到出结果平均
2.5 天
标本准备、细胞裂解--第
一天
酶切--第二天
染色、拍照--第三天
11
12
PFGE结果判读
目测法:
按美国疾病控制和预防中心(CDC)Tenover等人推荐的方
法判读。
图谱完全相同的定为一个型,彼此之间相差一个带的定
为同一型的不同亚型,相差2-3个带的认为亲缘关系密
切,相差4-6个带的认为可能相关,条带相差7个以上的
认为无亲缘关系。并随机地选择不同的字母如A、B、C、
D等的字母顺序分型。
聚类分析
计算机输入SPSS,做树状图
13
PFGE同源性分析
D ic e ( O p t:1 .5 0 % ) ( T o l 1 .5 % - 1 .5 % ) ( H > 0 .0 % S > 0 .0 % ) [0 .0 % - 1 0 0 .0 % ]
O X AP C R
C T XP C R
P C R T e s tin g
SH V PC R
PFGE - XbaI
TE MP C R
100
80
60
PF GE - XbaI
. E X .0 0 4 2
E
01 A
. E X .0 0 4 4
E
07 A
. E X .0 0 4 5
E
07 A
. E X .0 0 3 8
E
01 A
. E X .0 0 3 8
E
01 A
. E X .0 0 3 8
E
01 A
. E X .0 0 1 3
E
01 A
. E X .0 0 4 0
E
01 A
. E X .0 0 1 5
E
01 A
14
PFGE特点:
DNA原位提取法,减少了断裂
利用细菌全基因组信息
细菌分型金标准
15
2、RAPD(随机扩增DNA多态性)
1990年
Williams:
RAPD
Welsh:AP-PCR
本质上相同
引物:
“短” 、“单一” 和
“非特异性”,一般9~
10bp
扩增条件:“非严格性”
退火温度一般较低,25~
35℃
16
ESHWAR MAHENTHIRALINGAM.et al.J Clin
Microbiol,1996;34(5):1129~1135
17
ESHWAR MAHENTHIRALINGAM.et al.J Clin
Microbiol,1996;34(5):1129~1135
18
ESHWAR MAHENTHIRALINGAM.et al.J Clin
Microbiol,1996;34(5):1129~1135
19
分子分型在医院感染控制中
的作用
20
Special Issue
New Technology for Detecting MultidrugResistant Pathogens in the Clinical
Microbiology Laboratory
Lance R. Peterson*† and Gary A. Noskin*†
*Northwestern Memorial Hospital and
†Northwestern University Medical School,
Chicago, Illinois, USA
EID, 2001,
7: 30621
Northwestern Memorial Hospital,
Chicago
700-bed, university-affiliated medical
center
出院:>39,000/年
急诊:56,000例/年
门诊量:260,000/年
22
分型确认后及干预效果
5.79
P=0.002
LR Peterson
et al, EID,
2001, 23
7: 306
指标
1. the total number of nosocomial infections
per 1,000 patient days每千住院日医院感染
数
2. the number of patients with nosocomial
infections per 100 patient discharges
每100出院病人医院感染病人数
(percentage of patients with nosocomial
infection)
(医院感染病人百分比)
24
感染控制工作小组成员:
We formed a permanent, integrated infection
control and prevention program that fully
incorporates
infection control personnel,
infectious disease personnel,
pharmacy personnel,
clinical microbiology
personnel
into a single working group to
minimize hospital infections.
感染控制
感染性疾病
药学
临床微生物
Hacek DM et al. Am J Clin Pathol 1999;111:647-54.
25
资料收集方法(Methods for data
collection):
review of microbiology reports
review of patients' medical
records,
direct observation of medical
and nursing practice,
active surveillance of rectal
cultures of patients in nursing
units for high-risk patients,
evaluation of suspected
nosocomial infections reported
by health-care providers.
查阅微生物报告
查阅病史记录
对医护人员操作的直接
观察
高危病人直肠培养的动
态连续观察
对上报的可疑医院感染
的评估鉴定
由三位专职感染控制人员对资料进行汇总分析,并制
定出相应控制措施,并在医院感染控制和预防部门主管的指
导下实施。
26
Two interventions:
a molecular typing
laboratory
a weekly planning
meeting
infection control
diagnostic medical
microbiology (molecular
epidemiology)
Pharmacy
and infectious diseases
两个主要的干预措施:
分子分型
周会
包括以下方面的代表:
感染控制
微生物诊断(分子
流行病学)
药学
感染性疾病
27
weekly meetings:
周会内容:
the ongoing short- and long 医院感染动向(短
term trends in nosocomial
期、长期)
infections within the center
activities of the infection control 感染控制专职人员
professionals and microbiology
和微生物实验室的
laboratory personnel;
工作
any needed changes were
determined.
决定需要做的调整
The organizational structure for 需分型的病原体与
selecting microbes for typing
主管讨论决定
was shared by the medical
directors of infection control and
clinical microbiology
28
需基因分型的微生物:
routinely genomically typed:VRE
Periodic routine typing:
fluoroquinolone-resistant P.
aeruginosa,
methicillin-resistant Staphylococcus
aureus (MRSA),
Enterobacter cloacae,
Clostridium difficile
Additional organisms for typing:
selected through surveillance of
microbiology culture reports
discussed at the weekly meeting.
常规基因分型:VRE
周期性分型:
氟喹诺酮耐药的铜绿
MRSA
阴沟肠杆菌
难辨梭菌
其他:根据微生物报告
并在周会上讨论后决定
需基因分型的病原体
任何时候工作小组要求进行基因分型,临床微生物
29
实验室即将菌株提供给分子分型部门并执行
实验方法
REA analysis
restriction of genomic DNA with
conventional electrophoresis
DNA限制性酶切
30
成本-效益分析(Analysis of Cost Data)
基因分型相关费用
the cost of equipment,
remodeling,
reagent and other
supplies,
salaries and benefits
for three technologists,
plus all the institutional
assessments (e.g., fullcost basis) required to
operate a hospital
laboratory.
仪器配制及维修、实
验室改造等;
试剂及其他消耗品
三位实验员的薪水
所有的评估分析(如
full-cost basis )需
要动用全院系统
分析方法:t检验
31
结果:
1、VRE
initial impetus: serious
nosocomial problem--VRE's emergence
molecular typing results:
a pattern of numerous
“mini” patient-to-patient
outbreaks of distinct
clones
rather than the spread of
a single persisting strain
1、VRE
最初调查:VRE医
院感染严重
分型结果提示:
多型别、小规模
(mini)病人间流
行
而不是一个型别的
流行
32
结果:
genomic typing:
基因分型:可将可能的医院
patient-to-patient
transmission;
nosocomial outbreak;
little evidence of
horizontal spread
Using this information, we
determined what
intervention was likely to
control an apparent
outbreak (20).
感染分组:
病人之间交叉感染(high
conality, >90%)
感染爆发(moderate
clonality, 35%-75%)
无水平传播(<20%
clonality).
在此基础上,决定采取哪种
控制措施
33
high
conality,
>90%
moderate
clonality,
35%-75%
clonality,
<20%
likely
possibly
unlikely
patient-topatient
transmission
nosocomial
outbreak
little evidence
of horizontal
spread
similarity
34
During the last 2
years of this study,
25 possible microbial
outbreaks were
investigated by the
typing laboratory
VRE,
fluoroquinoloneresistant P. aeruginosa,
MRSA,
E. cloacae,
C. difficile.
通过基因分型,共鉴别
25起微生物感染爆发
VRE
氟喹诺酮耐药的铜绿
MRSA
阴沟
难辩梭菌
35
Classic Spread of Nosocomial 典型的医院感染传播
Infection
VRE: 19 strains, from 16 patients, in
a 2-month period;
14 strains: from one of two clones
(88%)
Indicating: a high probability of
nosocomial spread
Review:
检查分析:
microbiology laboratory: culture
requisitions---no close contact.
Patients: existing direct connection
between 11/14 patients (14).
infection control practices:
aborted the outbreak
VRE: 19株, 来自16个病
人,2个月时间内;
其中十四株:为两个型
别中的一个型别 (88%)
高度提示感染传播
微生物实验室: 培养过
程无密切联系
患者:14人中有11人有
直接联系
感染控制:中止暴发
36
Moderate Likelihood of Spread of
Nosocomial Infections
During a 1-month period, in a special- 较有可能为NI传播
1个月时间内,特殊病房
care unit
invasive infections, caused by five 侵入性操作感染:
肺炎克雷伯菌
isolates
表皮葡萄球菌
Klebsiella pneumoniae,
溶血葡萄球菌
S. epidermidis, and
40%-60% clonality
S. hemolyticus
DNA typing indicated 40% to 60% for 分析:分离出相同型别菌株的
each of the bacterial species.
patients with genetically identical
organisms occupied adjacent beds.
Erecting a barrier on the unit, along
with educating medical staff, halted
the spread of these infections (15).
患者病床临近
措施:
病房设立屏障
医护人员教育
结果:感染中止
37
Outbreaks not Caused by Patient-toPatient Spread
Suspected outbreaks
consisting of four isolates of K.
pneumonia and 64 strains of
Serratia marcescens were
investigated in the ICUs of two
hospitals. Both investigations
showed 21% clonality,
indicating unlikely patient-topatient spread.
Investigation suggested
suboptimal handling of
ventilator equipment, and both
outbreaks were stopped by
retraining of personnel using
this equipment
2个医院的ICU病房
4株肺克,64株粘质沙雷
菌
21% clonality
提示:非病人之间传播
indicating unlikely
patient-to-patient
spread.
调查分析:机械通气相关
操作不规范
措施:规范操作
结果:感染中止
38
Pseudooutbreaks
Possible outbreaks occurred in the
2个医院,特殊护理病
special-care nursery units of two
hospitals, each of which had its own
molecular typing section.
seven S. aureus strains, and the other
of four isolates of gram-negative bacilli.
immediately typed and no (20%)
clonality existed.
No interventions were instituted, and
the apparent outbreaks were
determined to be normal variation in
infections (15,21).
avoided culture-based surveillance
investigation of staff by the state
department of health, and the other
avoided closing the unit for a 30-day
full disinfection and cleaning (done in
previous suspected outbreaks).
房,每个医院都有自
己的分子分型部门
7株金葡 4株G-菌
迅速分子分型rapid
typing
no (20%) clonality
没有采取措施
节省:
医护人员携菌情况
调查(培养)
关闭病房30天,消
毒、清洁
39
分型确认后及干预效果
4
Typing Intervention
3.5
3.3
3
2.56
2.5
NI/100 Patients
2
1.5
1
0.5
P=0.000006
0
1993
1994
1995
1996
1997
1998
1999
LR Peterson
et al, EID,
2001, 40
7: 306
分型确认后及干预效果
5.79
P=0.002
LR Peterson
et al, EID,
2001, 41
7: 306
nosocomial infection:
3.3%-2.6% (national rate:
4.4%-5%)
>1,400 fewer patients
acquired infections
during this time,
averting more than 50
expected deaths
Even with endemic VRE,
most of our outbreaks
involve three or fewer
patients (19).
医院感染:3.3%下降
至2.6%(全国医院感染
率:4.4%-5%)
减少>1,400的病人感
染
死亡:减少了>50
VRE:涉及的病人也比
其他医院少
42
成本
The mean number of
医院感染患者数量平
patients with nosocomial
infections decreased by 283 均每年下降 283,住
per year, a reduction of
院天数下降超过1100
more than 1,100 inpatient
天
days.
The costs avoided by
因此节省的费用平均
using this calculation
averaged more than
$2,150,000/year, based on
1999 dollars.
每年超过$2,150,000
(与1999年相比)
43
Representatives now meet
together for 45 minutes
each week
For Microbiology, opening
the typing laboratory totaled
$180,050. By the fifth year,
costs in the laboratory
section were stable.
The cost for the laboratory,
includng three medical
technologists, is $400,000
yearly.
Virtually all these costs are
borne by the hospital.
小组聚会逐渐转为每
周开会,45min,讨
论
微生物室成立分子分
型实验室(设备及人
员)的费用为
$180,050.
每年分子分型相关支
出为$400,000
医院承担
44
While such a grant
program would cost up
to $2 billion each year
if all U.S. hospitals
participated,
the projected reduction
in cost of treating
nosocomial infections
could reach over five
times that amount.
a savings of $5.00 for
each dollar spent.
假设:美国所有医
院
进行基因分型相关
费用达到20亿美元
节省下来的治疗医
院感染的费用将超
过5倍(100亿)!
每使用1美元节省5
美元
45
Typing time: within 1 week
48 hours.
Lack of clonality: suggests
other reasons for the
apparent outbreak,
antimicrobial-agent use
pressure,
failure of appropriate nursingcare practices,
or simply random variation in
the number of infections.
时间:1周,48h
如没有流行相关线索,
可能是其他原因:
抗生素压力,
护理操作不当(非感
染相关),
仅仅是感染数量的随
机变化
Early knowledge of whether 早发现、及早确定调
microbial clonality is present
查范围、采取合适的
or absent focuses the scope
干预措施
of an investigation and
facilitates appropriate
intervention.
46
cost of rapid detection using
the polymerase chain reaction
(PCR)
=one day of glove isolation
could be completed in a single 8hour workday.
As gene chip technology
moves into clinical use,
detecting a large number of
resistance determinants soon
after a patient is admitted to
the hospital should be
possible.
PCR 分型
费用=一天的手套
费用
8小时的工作时间
内可完成
基因芯片:
大规模
耐药监测
病人入院后即实施
47
分子分型在医院感染中的应用:
technically possible
medically useful
economically justified
48
医院感染的分子流行病
学研究方法
分子流行病学方法在医
院感染中的应用
医院感染控制的人员安
临床医院感
染控制
科研
排
成本-效益研究
49
实验研究:
瑞金医院 04年~05年
全耐药鲍曼不动杆菌(PRAB) 各个科室的
突然增多
经脉冲场凝胶电泳(PFGE)证实
烧伤科为单独一个型别PRAB科室内流行
除烧伤科以外的其他科室为科室间同一型别
PRAB流行。
由此可见,分子流行病学方法:
为医院感染控制提供准确的实验数据
有效判断感染来源和流行趋势
为更好的做好医院感染控制工作打下了基础。
50